We wish to investigate ACTH activation of mammalian adrenal cortex cells to try to pinpoint molecular changes leading to the increased rate of the cytochrome P-450 dependent conversion of cholesterol into pregnenolone, the initial and rate limiting step of adrenocortical steroidogenesis. The principal emphasis of this research is to look for biophysical evidence of changes in phospholipid, steroid and protein composition and molecular organization and association, especially in adrenocortical mitochondrial membranes but also in the cytosol. We will also search for molecules that are newly synthesized or modified (for example by phosphorylation or methylation) in response to ACTH; the techniques used will be radioisotope labeling, gel electrophoresis, molecular sieving and ion exchange chromatography, high pressure liquid chromatography, etc. We will endeavor to detect physical changes, either global or local, in the mitochondrial mebranes. Examples would be: a change in membrane fluidity or in the distribution of molecules in the bilayer, changing the degree of association of the protein components of the cholesterol side chain cleavage enzyme system with each other or of the P-450 with its (membrane-soluble) substrate cholesterol. This would be pursued using EPR (particularly spin-label EPR) and fluorescence techniques to measure global changes in membrane fluidity and using reconstituted artificial membranes and/or micelles to try to mimic local changes in membrane fluidity or in local molecular organization. We believe that ACTH activation of mitochondria may be a highly complex process and we hope to assemble a coherent and correct picture.